Negative motor networks: Electric cortical stimulation and diffusion tensor imaging.

INTRODUCTION: This study investigated the networks of Negative motor areas (NMAs) using electric cortical stimulation and diffusion tensor imaging (DTI). METHODS: Twelve patients with intractable focal epilepsy, in which NMAs were identified by electrical cortical stimulation, were enrolled in this study. Electric stimulation at 50Hz was applied to the electrodes during motor tasks to identify the NMAs. DTI was used to identify the subcortical fibers originating from the NMAs found by electrical stimulation. RESULTS: NMAs were found in lateral frontal areas (premotor area (PM) and precentral gyrus) in all 12 patients, in pre-supplementary motor areas (pre-SMAs) in four patients, and in posterior parietal cortices (PPCs) in four. DTI detected fibers connecting to the ipsilateral PMs, PPCs and temporal regions via U-fibers, superior longitudinal fasciculus (SLF), and arcuate fasciculus (AF) from the lateral frontal NMAs. Pre-SMA-NMAs had connections with ipsilateral PMs and contralateral pre-SMAs via the frontal aslant tract and transcallosal commissural fibers, and PPC-NMAs with ipsilateral PMs via SLF and AF. CONCLUSION: This study found the characteristic cortical network of each NMA, and especially revealed new insight of pre-SMA-NMA and PPC NMA. These NMAs might be associated with different mechanism of negative motor response.

Missense variants of the alanine: glyoxylate aminotransferase 2 gene correlated with carotid atherosclerosis in the Japanese population.

Alanine:glyoxylate aminotransferase 2 (AGXT2; EC 2.6.1.44) degrades asymmetric dimethylarginine (ADMA), a competitive inhibitor of nitric oxide (NO) synthase. Increased ADMA, reduced NO, and hypertension are shown in Agxt2 knockout mice. There are four single nucleotide polymorphisms (rs37370, rs37369, rs180749, and rs16899974) with which AGXT2 activity changes in humans and may be related to vulnerability of vascular sclerosis. To examine the relationship between them, we studied the functional haplotypes of the AGXT2 gene and decided their relationship with arteriosclerotic changes via carotid intima-media thickness (carotid IMT) in Japanese subjects. Genotyping of those polymorphisms and the carotid IMT in 1,426 Japanese subjects were then evaluated. Subjects with C-A-A-A haplotype (rs37370, rs37369, rs180749, rs16899974) showed low AGXT2 activity (P<0.0001; Pearson’s correlation coefficients: 0.497). The C-A-A-A haplotype was significantly associated with mean carotid IMT (P=0.049) and max carotid IMT (P=0.004). Subjects with two C-A-A-A haplotypes exhibited thicker mean carotid IMT (P=0.022) and maximum carotid IMT (P=0.001). In multiple regression analysis, subjects with two C-A-A-A haplotypes were independently and positively associated with mean carotid IMT (P=0.02) and maximum IMT (P=0.005) after correction. There was a significant correlation between the functional variants in the AGXT2 gene and carotid IMT in Japanese. The AGXT2 genotype may be an important factor underlying atherosclerosis.

Effect of tyrosine kinase inhibition by imatinib mesylate on mast cell tumors in dogs.

BACKGROUND: Imatinib mesylate is a small molecule targeted at dysregulated protein-tyrosine kinase. Mutation of c-kit exon 11, which induces constitutive phosphorylation of KIT, is one of the mechanisms for the development or progression of mast cell tumor (MCT) in dogs. The purpose of this study was to examine the therapeutic potential of imatinib mesylate in canine MCT. HYPOTHESIS: Imatinib mesylate has activity against MCT in dogs, and response to treatment can be correlated to presence of mutation within exon 11 of c-kit. ANIMALS: Twenty-one dogs with MCT with gross tumor burden and median tumor size of 7.2 cm (range, 1.0-25.3 cm) before treatment. METHODS: Tumors were analyzed for mutation of c-kit exon 11. Imatinib mesylate was administered PO to the dogs at a dose of 10 mg/kg daily for 1-9 weeks. RESULTS: Ten of 21 dogs (48%) had some beneficial response to imatinib mesylate treatment within 14 days of treatment initiation. All 5 dogs with a demonstrable c-kit mutation in exon 11 responded to the drug (1 complete remission, 4 partial remission). CONCLUSIONS AND CLINICAL IMPORTANCE: Imatinib mesylate has clinical activity against MCT in dogs. Response could not be predicted based on presence of absence of a mutation in exon 11 of c-kit.

Fate of nonylphenol and 17beta-estradiol contained in composted sewage sludge after land application.

Many environmental problems caused by endocrine disrupters (EDs) have been reported. Because little is known about the fate of EDs accumulated in sewage sludge, we carried out a study to clarify the fate of EDs in composted sludge after its application to soil. Nonylphenol (NP) and 17beta-estradiol (E2) were measured for leachate and soil. High concentrations of NP and E2 were detected in the leachate at the early stage, but they decreased rapidly. Also, the high contents of NP and E2 in soil decreased significantly within 300 days. Because the decrease of NP and E2 in the soil was much larger than that of NP and E2 in the leachate, there must have been a physicochemical or biological decomposition mechanism in the soil layer. We also tried to clarify the transfer of NPs to plants from compost. In the experimental conditions of this study, the transfer of NPs to plants from compost was not observed.

Fate of nonylphenol polyethoxylates and nonylphenoxy acetic acids in an anaerobic digestion process for sewage sludge treatment.

Many environmental problems caused by endocrine disruptors (EDs) have been reported. It is reported that EDs flow into sewage treatment plants, and it has been pointed out that these may be shifted from the wastewater treatment process to the sludge treatment process. Little is known about the fate of EDs accumulated in sewage sludge, so we carried out a study to clarify the fate of EDs in sewage sludge treatment processes, especially in an anaerobic digestion process. In this study, nonylphenol (NP) was selected as a target ED. Nonylphenol ethoxylates (NPnEO) or nonylphenoxy acetic acids (NPnEC), which were the precursor of NP, were added to an anaerobic digestion process, and mass balance was investigated. The following results were obtained from the anaerobic digestion experiments. (1) NP1EO was injected to an anaerobic digestion testing apparatus that was operated at a retention time of approximately 28 d and a temperature of 35 degrees C with thickened sludge sampled from an actual wastewater treatment plant. Approximately 40% of the injected NP1EO was converted to NP. (2) NP1EC was injected to an anaerobic digestion testing apparatus with thickened sludge. As a result, almost all injected NP1EC was converted to NP. When NP2EC was injected, NP2EC was not converted to NP until the 20th day.

Clostridium perfringens beta-toxin is sensitive to thiol-group modification but does not require a thiol group for lethal activity.

The beta-toxin gene isolated from Clostridium perfringens type B was expressed as a glutathione S-transferase (GST) fusion gene in Escherichia coli. The purified GST-beta-toxin fusion protein from the E. coli transformant cells was not lethal. The N-terminal amino acid sequence of the recombinant beta-toxin (r toxin) isolated by thrombin cleavage of the fusion protein was G-S-N-D-I-G-K-T-T-T. Biological activities and molecular mass of r toxin were indistinguishable from those of native beta-toxin (n toxin) purified from C. perfringens type C. Replacement of Cys-265 with alanine or serine by site-directed mutagenesis resulted in little loss of the activity. Treatment of C265A with N-ethylmaleimide (NEM), which inactivated lethal activity of r toxin and n toxin, led to no loss of the activity. The substitution of tyrosine or histidine for Cys-265 significantly diminished lethal activity. In addition, treatment of C265H with ethoxyformic anhydride which specifically modifies histidyl residue resulted in significant decrease in lethal activity, but that of r toxin with the agent did not. These results showed that replacement of the cysteine residue at position 265 with amino acids with large size of side chain or introduction of functional groups in the position resulted in loss of lethal activity of the toxin. Replacement of Tyr-266, Leu-268 or Trp-275 resulted in complete loss of lethal activity. Simultaneous administration of r toxin and W275A led to a decrease in lethal activity of beta-toxin. These observations suggest that the site essential for the activity is close to the cysteine residue.

Phosphoinositide turnover enhanced by angiotensin II in isolated rat glomeruli.

To clarify the signal transduction mechanism of angiotensin II in renal glomeruli, we studied the effect of the hormone on phospholipid metabolism using isolated rat glomeruli. Stimulation of the glomeruli pulse-chase labeled with [3H]glycerol by angiotensin II caused a rapid (within 15 s) breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) with a concurrent production of 1,2-diacylglycerol. This effect of angiotensin II was in a dose-dependent manner within the range from 10(-12) M to 10(-6) M, and was inhibited by saralasin. Angiotensin II also decreased the 3H radioactivity of PIP slightly only at 15 s and increased that of phosphatidic acid after 15 s, with no significant effect upon the labelings of phosphatidylinositol (PI), phosphatidylcholine (PC) and phosphatidylethanolamine (PE) within 1 min. The change in phospholipid metabolism by angiotensin II was similar when the glomeruli were labeled with [32P]orthophosphate: the decrease in the labeling of PIP2 and the increase in the labeling of phosphatidic acid after 15 s. In addition, 32P labeling of PI increased after 2 min. These results suggest that angiotensin II, after binding to glomerular receptors, induces initial PIP2 hydrolysis to diacylglycerol and subsequent resynthesis of PIP2 through phosphoinositide turnover.

Favorable outcomes with tacrolimus in two patients with refractory interstitial lung disease associated with polymyositis/dermatomyositis.

Two cases of progressive interstitial lung disease associated with polymyositis/dermatomyositis are presented. Both patients were refractory to conventional therapy with high-dose corticosteroids, cyclosporine, and intermittent pulse cyclophosphamide, and thus a therapeutic trial of tacrolimus was instituted. Tacrolimus was markedly effective in achieving subjective, laboratory and radiographic improvement in both patients.

Calcium-activated, phospholipid-dependent protein kinase in cultured rat mesangial cells.

The analysis of the 100 000 X g supernatant fraction of cultured rat glomerular mesangial cells with DEAE-cellulose ion-exchange chromatography revealed a large peak showing the activity of a protein kinase (protein kinase C) which depended on phospholipid and diolein as well as Ca2+. Furthermore, it was shown that angiotensin II (AII) (10(-6)M) induced rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate, leading to production of diacylglycerol rich in arachidonic acid, in the cultured rat mesangial cells. These results suggest that activation of protein kinase C resulting from enhancement of phosphoinositide metabolism may be important as an intracellular regulatory mechanism of AII upon cultured mesangial cells.

Signal transduction mechanism of interleukin 6 in cultured rat mesangial cells.

Interleukin 6 (IL-6) is one of the potent autocrine growth factors for mesangial cells. We investigated the signal transduction mechanism of IL-6 in cultured rat mesangial cells. IL-6 induced a transient increase of inositol 1,4,5-trisphosphate (Ins 1,4,5-P3) followed by a transient and sustained increase of intracellular calcium concentration, suggesting that IL-6 stimulates phosphoinositide turnover. IL-6 also stimulated prostaglandin E2 (PGE2) production. The IL-6-concentration dependency in PGE2 production was similar to that in Ins 1,4,5-P3 production. We concluded that the action of IL-6 on mesangial cells is exerted at least partially through the enhancement of phosphoinositide turnover and PGE2 production.

Endothelin-1 stimulates prostaglandin E2 production in an extracellular calcium-independent manner in cultured rat mesangial cells.

To study a possible role of endothelin-1 (ET-1) in the regulation of glomerular function, we examined the presence of receptors for, and the biological action of, ET-1 in cultured rat mesangial (M) cells. The first-subcultured M cells prepared from isolated glomeruli of Sprague-Dawley rats were used. ET-1 binding was assayed by using 125I-ET-1. Inositol 1,4,5-trisphosphate (IP3) was determined by IP3-specific binding assay. Intracellular calcium (iCa2+) was measured in fura-2 loaded cells. Prostaglandin E2 (PGE2) was measured by radioimmunoassay. In M cells there existed two classes of binding sites specific for ET-1 (Kd was 0.24 and 4.4 nmol/L, Bmax was 130 and 1070 fmol/mg, respectively). ET-1 (10(-7) mol/L) induced a rapid and transient increase in IP3, followed by transient and sustained increases in iCa2+. Nicardipine (10(-6) mol/L) inhibited only the sustained increase in iCa2+. ET-1 (10(-9) mol/L to 10(-7) mol/L) significantly stimulated PGE2 production with the concentration dependency. Nicardipine (10(-6) mol/L) and diltiazem (10(-6) mol/L) did not inhibit the PGE2 production. We conclude that M cells have specific ET-1 receptors linked to phosphoinositide turnover and PGE2 production, and PGE2 production by ET-1 may be through an extra-cellular calcium-independent mechanism. Our results suggest that ET-1 plays an important role in the regulation of glomerular functions by modulating PGE2 production in M cells.

Efficacy of oral and systemic antibiotic prophylaxis in colorectal operations.

A cooperative Veterans Administration study of the septic complication rate during large-bowel surgery was undertaken in two groups of patients. The first group received oral neomycin and erythromycin base plus parenteral placebo; the second, the oral antibiotics plus parenteral cephalothin sodium. During a five-year period, 1,128 patients were studied. The overall septic complication rate was 7.8% in patients receiving only oral antibiotics, and 5.7% in patients receiving both oral and parenteral antibiotics. This difference was not significant. The only significant finding was a greater incidence of fever of unknown origin in patients receiving only oral antibiotics. None of those patients were treated with additional antibiotics, and all fevers cleared spontaneously. There seems to be no discernible benefit from adding parenteral antibiotic prophylaxis when performing elective colon surgery if appropriate mechanical cleansing and oral neomycin and erythromycin therapy are employed.

Enzyme-linked immunosorbent assay for rapid detection of toxins from Clostridium perfringens.

An enzyme-linked immunosorbent assay (ELISA) with antibodies specific to beta, epsilon and iota ib toxins of Clostridium perfringens was developed to detect beta, epsilon and iota ib toxins, respectively. The ELISA was sensitive enough to detect as little as 1.0 ng/ml of purified beta and iota ib toxins and 0.1 ng/ml of purified epsilon toxin. By means of the ELISA method, 192 isolates of C. perfringens from food samples in Japan and Thailand, and 58 isolates from patients suffering from gas gangrene or gastroenteritis were examined. One isolate from food samples in Japan, three from food samples in Thailand and five from stools of patients with gastroenteritis were C. perfringens type D. One type B and one type C were detected from the stools of patients with gastroenteritis.

Effects of fluorocarbon infusion on microcirculation during cardiopulmonary bypass.

A pulsatile pump was used to effect a three-hour cardiopulmonary bypass in 15 mongrel dogs. During the bypass, Fluosol-DA (35%) infusion was used in lieu of homologous blood transfusions. The greater omentum was chosen as a model of extraorganic microcirculation, and its reaction to the infusion was observed. The omentum's microcirculation was not adversely affected by the Fluosol-DA (35%): no sludging, microthrombi, arteriovenous fistulas, plasma skimming, stasis, or other adverse findings were observed. The animals' oxygen consumption did not decrease, and the resistance of the systemic blood vessels, did not increase during the experiment, suggesting that the fluorocarbon infusion had no untoward effects on the microcirculation.

Partial agonist activity of celiprolol, a cardioselective beta-antagonist.

The beta-antagonistic effects of celiprolol were assessed in isolated guinea-pig preparations. The pA2 values obtained were 7.84 +/- 0.07, 7.79 +/- 0.06 and 6.45 +/- 0.11 against the positive chronotropic and inotropic effects in the atria and relaxant effects in the trachea induced by isoproterenol, respectively, indicating that celiprolol is a cardioselective beta-antagonist. Celiprolol produced slight positive chronotropic and inotropic effects in atrial preparations and a uniquely strong relaxant effect in tracheal preparations. All these effects were attenuated by pretreatment with 10(-6) M atenolol or 10(-6) M propranolol. In pithed rats, celiprolol produced pressor responses at doses lower than 0.03 mg/kg and hypotensive responses at doses higher than 0.3 mg/kg. It produced sustained increases in heart rate in all doses applied. These effects were attenuated by pretreatment with 1 mg/kg of propranolol. The pressor response might be taken to be a reflection of an increase in cardiac function mediated by cardiac beta 1-receptor stimulation, while the hypotension may be ascribed mainly to stimulation of beta 2-adrenoceptors in the vascular system. It is concluded that celiprolol exerts a uniquely strong partial agonist activity at beta 2-adrenoceptors.

Asymptomatic membranous obstruction of the inferior vena cava forming intrahepatic collateral pathways.

Intrahepatic and/or extrahepatic collateral pathways result from the membranous obstruction of the inferior vena cava. These collaterals are usually insufficient to prevent Budd-Chiari syndrome. We reprot an unusual case of asymptomatic membranous obstruction of the inferior vena cava in which marked intrahepatic collateral pathways were formed. Although the inferior vena cava terminated above the orifice of the right hepatic vein, the middle and left hepatic veins were patent above the membrane, without narrowing. Blood from the inferior vena cava drained into the right atrium via the intrahepatic collaterals between the right and middle hepatic veins without resistance.

Preoperative prophylactic cephalothin fails to control septic complications of colorectal operations: results of controlled clinical trial. A Veterans Administration cooperative study.

Data obtained from a survey of the membership of the Society for Surgery of the Alimentary Tract and the American Society of Colon and Rectal Surgeons indicated that concomitant administration of oral neomycin-erythromycin base and systemic cephalothin, together with mechanical colon cleansing, was the most popular method of colon preparation. We designed a prospective double blind clinical trial to compare administration of intravenous cephalothin, oral neomycin-erythromycin base, and the combination of both the intravenous and oral antibiotics. Intake of patients to the intravenous cephalothin group was stopped because the data indicated that this method of prophylaxis resulted in significantly higher numbers of septic complications. The incidence of wound infection was 30 per cent and the overall incidence of septic complications was 39 per cent in patients receiving only intravenous cephalothin combined with mechanical colon cleansing. The incidence of wound infection and the overall incidence of septic complications was only 6 per cent in the comparison group, and the differences are highly significant.

Factors affecting implant mobility at placement and integration of mobile implants at uncovering.

This study examined 1) factors that contributed to implant stability at placement and 2) the likelihood for an implant that was mobile at placement to osseointegrate. Eighty-one (3.1%) of 2,641 implants placed by the Dental Implant Clinical Research Group between 1991 and 1995 were found to be mobile at placement. Seventy-six (93.8%) of the 81 mobile implants were integrated at uncovering compared to 97.5% for the 2,560 immobile implants. Variables that influenced mobility at placement included patient age, implant design and material, anterior-posterior jaw location, bone density, and use of a bone tap. Hydroxyapatite (HA)-coated implants were slightly more likely to be mobile at placement (P = 0.324) than non-hydroxypatite (HA)-coated implants. Of the 54 HA-coated implants that were mobile at placement, all (100%) integrated, while only 17 (81.5%) of the 22 mobile non-HA-coated implants integrated (P = 0.003). Mean electronic mobility testing device values (PTVs) at uncovering for all implants mobile or immobile at placement that integrated were -2.9 and -3.6 respectively. PTVs for HA-coated implants that were mobile (-3.5 PTV) or immobile (-4.0 PTV) at placement differed by 0.5 PTV, whereas non-HA-coated implants exhibited a greater difference of 1.2 PTVs at uncovering. HA-coated implants, regardless of mobility at placement, integrated more frequently and exhibited greater stability than non HA-coated implants.

Characterization of bacteriocin N15 produced by Enterococcus faecium N15 and cloning of the related genes.

Enterococcus faecium N15 was isolated from nuka (Japanese rice-bran paste), which is utilized as starter in the fermenting of vegetables, and was found to produce a bacteriocin that exhibited a broad spectrum of activity, including activity against Listeria monocytogenes and Bacillus circulans JCM2504. The bacteriocin was sensitive to proteases (alpha-chymotrypsin, proteinase K, trypsin, and pepsin) and alpha-amylase, but it was resistant to lipase. The bacteriocin was resistant to heat treatment at 100 degrees C for 2 h, but its activity was completely lost after autoclaving at 121 degrees C for 15 min. It was active over a wide pH range from 2.0 to 10.0. The bacteriocin showed bactericidal activity against Lactobacillus sake JCM1157 at a concentration of 40 AU/ml. Its molecular weight was estimated by SDS-PAGE to be about 3-5 kDa. PCR primers were designed based on the conserved amino acid sequences of class IIa bacteriocins. A 3-kb DNA fragment was amplified and three open reading frames (ORFs) were found. The first encodes a probable immunity protein of 103 amino acid residues and shows complete homology with the putative immunity protein of E. faecium DPC1146. The second and third ORFs respectively encode a probable transposase gene and an inducing factor. The upstream region of the immunity gene, in which the bacteriocin structural gene is located, was amplified. A homology search revealed that the bacteriocin produced by E. faecium N15 exhibits complete identity to enterocin A, a bacteriocin produced by E. faecium DPC1146. PCR using the primers designed in this study is a rapid and sufficient method of screening for bacteriocin-producing strains.